Amyotrophic Lateral Sclerosis (ALS) is a devastating disease in which progressive degeneration of motor neurons leads to paralysis, usually resulting in death 2-5 years after diagnosis. No therapies exist that significantly increase quality of life or life expectancy. Mutations in >30 genes are linked to ALS onset, albeit >90% of all ALS cases are sporadic. However, a unifying cellular hallmark in >95% of all ALS cases is the cytoplasmic mis-localization, accumulation, and aggregation of the nuclear RNA binding protein TDP-43 (TAR DNA/RNA binding protein 43) in motor neurons and support cells. Similar TDP-43 pathology is observed in other neurodegenerative diseases, including in forebrain neurons of ~50% of Frontotemporal dementia patients (FTD). TDP-43 pathology confers a toxicity to neurons, but the nature of this toxicity remains fiercely debated. Loss of nuclear function (LOF) and gain of cytoplasmic function (GOF) mechanisms have been proposed, though separating such mechanisms and identifying the earliest impacts of TDP-43 pathology has remained elusive. Regardless, a therapeutic strategy that has shown promise in some ALS models is promoting the degradation of cytoplasmic TDP-43. Recently, cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomal pathway, which, when induced, suppresses TDP-43 toxicity. However, understanding of this degradation pathway remains limited. Key gaps in understanding include determining, in an ALS-relevant neuronal model, the earliest and most disease-relevant impacts of TDP-43 pathology and defining how endolysosomal TDP-43 degradation occurs. The aims of this grant are: 1.) Establish a novel endogenous TDP-43 reporter system in neurons that allows precise control of TDP-43 abundance and cellular localization via small molecule and optogenetic means. Using this system, the impacts of altered TDP-43 levels and localization on TDP-43 itself, ALS phenotypes and gene expression, focusing on RNA abundance and translation, will be examined. 2.) Test an endolysosomal degradation model involving TDP-43 ubiquitination and endosomal membrane invagination in neurons using an optical pulse labelling approach. TDP-43 degradation mechanisms will also be defined in patient-derived ALS models using similar means. Finally, a novel high throughput yeast dot-blot assay will be used to identify genetic and chemical regulators of TDP-43 and Fused in Sarcoma (FUS) abundance, which is also implicated in ALS and FTD pathology. This grant is innovative in that a novel approach to exert spatiotemporal control of TDP-43 expression, which promises separation of TDP43 LOF and GOF toxicity effects, and a means to identify regulators of TDP-43 and FUS abundance via dot blot, are proposed. Finally, mechanistically defining endolysosomal-based means of cytoplasmic TDP-43 degradation promises new basic insight into proteostasis for TDP-43 and other substrates. In summary, TDP-43 and FUS are logical entry points for the study of ALS...